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| Home > Publications database > Heterologe Expression, Kristallisation und Untersuchungen zur Struktur von Bos taurus $\beta$-Arrestin und Rattus norvegicus PAR-4 |
| Home > Publications database > Heterologe Expression, Kristallisation und Untersuchungen zur Struktur von Bos taurus $\beta$-Arrestin und Rattus norvegicus PAR-4 |
| Book/Report | FZJ-2020-00827 |
2001
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/24167
Report No.: Juel-3876
Abstract: $\beta$-$\textbf{arrestins}$ are essential regulators of G-Protein coupled receptors. In addition to terminatingthe signal cascade, they mediate the endocytosis of the receptors through "clathrin coatedpits." In this work the protocols for the heterologous expression and purification of bovine$\beta$-arrestin in $\textit{S.cerevisiae}$ are established. Through mutagenesis affinity tags were added to the$\beta$-arrestin gene. Affinity chromatography yielded up to 2.7 mg of protein per gram of cells.The purity of the protein was optimized by usage of two different affinity tags (C- and Nterminus)and sequential purification. The stability against oxidation and the solubility of theprotein were significantly increased through the mutagenesis of four cysteine residues toserines and the employment of high concentrations of phosphate.The functionality of the heterologously expressed $\beta$-arrestin was demonstrated by means of itscapacity to bind rhodopsin. Using tartrate as a precipitant, crystals of purified protein weregrown as large as 800 pm; however the low stability and poor diffraction hamperedcrystallographic success. By co-crystallization of $\beta$-arrestin with its ligand inositolhexaphosphate(IP$_{6}$) the diffraction quality of the crystals was dramatically improved. Neitherthe type nor the position of the affinity tag used had any significant influence on theappearance of the crystals hence on the packing of the molecules. Crystallographic data setsobtained using synchrotron radiation are currently being analyzed to solve the three-dimensionalstructure of $\beta$-arrestin by molecular replacement of the known structure of visualarrestin. Furthermore co-crystallized heavy metal derivatives of $\beta$-arrestin were performed,thereby enabling structure-determination independent of the arrestin-model by means ofisomorphous replacement.A first result to emerge from this analysis was the mapping of the IP$_{6}$-binding site to the C-terminaldome of the $\beta$-arrestin molecule as previously proposed. Furthermore there was noevidence in support of additional binding-sites for IP$_{6}$ as postulated in the literature.Further work will involve the improvement of the diffraction quality of the crystals,the determination of the three-dimensional structure of $\beta$-arrestin as well as attempts toco-crystallize $\beta$-arrestin together with other proteins which interact specifically with it.$\textbf{PAR-4}$ plays an essential role mediating the susceptibility of cells towards apoptosis, is activein certain tumors and is a protagonist responsible for some symptoms of variousneurodegenerative diseases such as the Alzheimer's or Parkinson's. In the apoptotic signalcascade PAR-4 seems to act at an early stage.With the experiments presented in this thesis attempts were made to heterologously expressand to crystallize PAR-4. First trials failed due to the low solubility of the protein, a problemwhich was overcome with high concentrations of divalent cations. Using affinity tags andchromatographic methods sufficient amount of highly purified protein, suitable forcrystallization were obtained from E.coli (10 mg/g cells) as well as from $\textit{S.cerevisiae}$ (2 mg/gcells). Functionality of the protein on the physiological level was tested with a kinase assay.The structure of the isolated protein and its integrity was investigated and preliminarilydescribed by biophysical techniques such as NMR and FTIR-spectroscopy. Crystallizationattempt so far have yielded crystals unsuitable for crystallographic pursuits. These resultsserve as groundwork towards the three dimensional structure of PAR-4.
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